96 well

The antifungal effect of Pom-1A to Pom-1F on Candida spp. biofilm formation can be determined following the Clinical and Laboratory Standard Institute guidelines (M27-A3) [67 ]. For this, 2.5 × 10³ yeast cells were incubated in 200 µL of RPMI-1640 medium supplemented with L-glutamine, fluconazole, amphotericin B, and Pom-1A to Pom-1F. Incubation was performed on flat-bottomed polystyrene microplates with 96 wells (Sarstedt AG & Co. KG, Nümbrecht, Germany) for 24 h at 37 °C without shaking. The subsequent treatment with crystal violet was originally developed by George O’Toole for bacteria and adapted to Candida biofilms [68 (link)]. For this purpose, the planktonic phase was removed, and the wells were washed twice with 200 µL of demineralized water. The remaining biofilm cells were treated with 200 µL of 0.1% (w/v) crystal violet solution for 15 min. After removing the solution, they were washed again twice with 200 µL demineralized water, and the microtiter plates were dried for at least 24 h at 25 °C. The stain was dissolved with 200 µL of 30% acetic acid and transferred to a new plate after 15 min. The absorbance at 560 nm was measured using a Tecan Infinite F200 microplate reader (Tecan Group Ltd., Männedorf, Switzerland). The resulting data were evaluated against the untreated controls so that the efficacy of the agents could be determined.

A resazurin assay was used to detect the antifungal effect of Angicin on the viability of Candida cells. Therefore, 2.5 × 10³ cells were incubated in 200 μl RPMI-1640 medium with 300 mg/l L-glutamine and two different Angicin concentrations (25 μg/ml, 100 μg/ml) at 37°C for 24 h in a flat-bottomed polystyrene microtiter plate with 96 wells (Sarstedt AG & Co., KG, Nümbrecht, Germany) with shaking at 900 rpm on an Eppendorf shaker. For the following quantification of viable cells, a resazurin-Reduction-Assay was performed. In brief, 20 μl of 0.15 mg/ml resazurin (Sigma-Aldrich) solution was added per well and incubated for 2 h at 37°C while shaking at 900 rpm. Fluorescence measurement (excitation wavelength 535 nm, emission wavelength 595 nm) of the resulting resorufin (viable cells are able to reduce resazurin to resorufin) was then performed by using a Tecan infinite F200 microplate reader (Tecan Group). The resulting data were normalized to the untreated control and the efficacy of Angicin was determined.

In vitro cytotoxic effects of PYDP on colorectal adenocarcinoma cell lines (HT‐29), breast cancer cell lines (MCF‐7) and mouse fibroblast (3T3‐L1) cell line were measured by the XTT method according to the manufacturer‘s instructions (Cell Proliferation XTT Kit, BI). The cells were seeded in 96‐well (Sarstedt, Numbrecht, Germany) at a density of 10,000 cells/well in a total volume of 100 μL. After 2 h of incubation (at 37 °C in a 95 % humidity and in an atmosphere containing 5 % CO₂), the medium was removed from the adherent cells and the wells were washed with 50 μL PBS buffer. Then, 50 μL of fresh medium was added per well followed by 50 μL of the sample (PYDP) at two concentrations (100–250 μM). After 24 h of incubation, the medium in each well was removed. Then, 100 μL of basal medium and 50 μL XTT solution were added to each well. To the control wells, 100 μL of complete medium containing 0.2 % DMSO was added. The plate was placed in a CO₂ incubator for 5–6 h. Finally, the absorbances at 450 nm (reference filter at 650 nm) were measured in an ELISA reader (Anthos‐HTII) and cell viability in the wells was calculated.